Project description:Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS gene encoding sacsin. ARSACS patients and mouse models display early degeneration of cerebellum in agreement with high sacsin expression in this organ. We performed unbiased transcriptomic of cerebella from Sacs KO mice versus controls to dissect the mechanisms underlying cerebellar degeneration in ARSACS.
Project description:The cerebellum harbors a circadian clock that can be shifted by scheduled mealtime and participates in behavioral anticipation of food access. To determine which cerebellar proteins are modified by time-of-day and/or feeding time, we determined day-night variations of proteome in the cerebellum of mice fed either ad libitum or only during daytime (from noon to lights off). Two-dimensional differences in gel electrophoresis (2D-DIGE) combined with two-way analyses of variance reveals that a majority of cerebellar proteins are significantly regulated by feeding conditions (food availability). Levels of few other cerebellar proteins were modulated exclusively by daily (or circadian) cues, independent of meal time, and others due to combined influence of meal time and time-of-day. Changes reflect behavioral anticipation of mealtime and/or feeding-induced shift in the circadian clock of the cerebellum.
Project description:DOT1L as methyltransferase of H3K79 is implicated in brian development. Here, we further defined DOT1L function in gene expression during cerebellar development using Microarrays. For that we generated Dot1l knockout mice using a Atoh-Cre driver line resulting in a Dot1l knockout within the cerebellum. The RNA of cerebellar tissue of the Dot1l knockout animals was thereby compared to controls. Additionally we compared the RNA levels of cultured CGNP and CGN samples treated with a DOT1L inhibitor versus DMSO treated cells. The data sets reveals potential new gene expression targets of DOT1L in vivo and in vitro, which ensure a correct development of the cerebellum.
Project description:DREAM (downstream regulatory element antagonist modulator) is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. Previous studies have shown a role for DREAM in cerebellar function regulating the expression of the sodium/calcium exchanger3 (NCX3) in cerebellar granules to control Ca2+ homeostasis and survival of these neurons. To achieve a more global view of the genes regulated by DREAM in the cerebellum, we performed a genome-wide analysis in transgenic cerebellum expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Our results indicate that DREAM is a major transcription factor in the cerebellum that regulates genes important for cerebellar development.
Project description:Neurons within the cerebellum form temporal-spatial connections through the cerebellum, and the entire brain. Organoid models provide an opportunity to model the early differentiation of the developing human cerebellum, which is difficult to study in vivo, and affords the opportunity to study neurodegenerative and neurodevelopmental diseases of the cerebellum. Previous cerebellar organoid models focused on early neuron generation and single cell activity. Here, we modify previous protocols to generate more mature cerebellar organoids that allow for the establishment of several classes of mature neurons during cerebellar differentiation and development, including the establishment of neural networks during whole organoid maturation. This will provide a means to study the generation of several more mature cerebellar cell types, including Purkinje cells, granule cells, interneurons expression as well as neuronal communication for biomedical, clinical, and pharmaceutical application.
Project description:The U2 snRNA is a basal component of the major spliceosome, which is responsible for >90% human pre-mRNA splicing. A 5-nucleotide deletion in one of the mouse U2 snRNA genes (Rnu2-8) causes cerebellar granule cell degeneration in the NMF291 mouse mutant strain. To identify the altered transcripts in the NMF291–/– cerebellum, we interograted Affy. mouse 1.0 ST Exon arrays with total RNAs from three postnatal-30-day (P30) wild type and three NMF291–/– cerebella.
Project description:The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated here in mice using large-scale two-dimensional differences in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24-h based on cosinor regression. Notably, rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only 6 proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including 2 proteins involved in synapse activity (Synapsin 2 (Syn2) and vesicle-fusing ATPase (Nsf)), 2 others participating in carbohydrate metabolism (triosephosphate isomerase (Tpi1) and alpha-enolase (Eno1)), and Glutamine synthetase (Glul), as well as Tubulin alpha (Tuba4a). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. This study highlights extensive rhythmic aspects of the cerebellar proteome.
Project description:Serial analysis of gene expression (SAGE) was used to identify and quantify all expressed cerebellar genes in the adult (P92; GSM17430) and aged (P810; GSM17226) C57BL/6J mouse cerebellum. A "closest-neighbor" algorithm was used to differentiate low abundance tags from possible sequencing errors in both libraries. Unique tags were categorized into four groups: (1) novel genes; (2) ESTs; (3) RIKEN, KIA, and hypothetical genes; and (4) known genes. Known genes were further subdivided into functional categories based on the gene ontology classification, using a web-based program developed in this laboratory (MmSAGEClass). Comparison of adult and aged cerebellar libraries revealed several genes that were differentially expressed, including growth hormone and prolactin, both of which were markedly decreased in the aged cerebellum. In addition, several tags showing differential expression were not identified in the Unigene database and are likely to represent novel genes. The present SAGE data on the aged cerebellar transcriptome may reveal candidate genes involved in the aging process. Keywords: other